USP18在干扰素-α治疗慢乙肝过程中的预测意义及作用机制

发布时间：2018-07-08 13:22

本文选题：乙型肝炎病毒 + 慢性乙型肝炎　；参考：《河北医科大学》2013年硕士论文

【摘要】：目的：乙型肝炎病毒（HBV）感染已经成为全球公共健康问题，每年约有100万人死于HBV感染相关的肝脏疾病。活动性HBV复制是导致肝脏损伤和疾病进展的关键因素，因此最大限度地长期抑制或清除HBV成为慢性乙型肝炎治疗的总体目标。目前用于慢性乙型肝炎抗病毒治疗的药物主要有两大类：1、核苷（酸）类似物；2、干扰素（interferon, IFN）。干扰素具有抗病毒和免疫调节双重作用，治疗疗程短而确定、耐药少见，故逐渐成为临床治疗慢性乙型肝炎的主要选择。然而，目前干扰素治疗只能达到30%-40%的HBeAg血清学转换，约有60%的患者尽管接受干扰素抗病毒治疗，但仍然遭受乙肝病毒的侵害，因此短期内预测干扰素治疗疗效显得尤为重要，最新研究热点是干扰素抗病毒作用信号通路中的干扰素刺激基因，泛素特异性蛋白酶18（Ubiquitin-specific protease18, USPl8）是其中重要的一个干扰素刺激基因。本研究旨在探讨USPl8在干扰素-α治疗慢乙肝过程中的预测意义及作用机制。方法：选取慢性HBV感染者70例，均为免疫清除期患者，所有患者均进行肝组织活检，无明显干扰素抗病毒治疗禁忌症。留取受试者治疗前新鲜外周血提取PBMCs进行体外干扰素-α孵育刺激试验，并对受试者应用干扰素-α抗病毒治疗，进行随访观察24周，其中无病毒学应答患者（nonvirological response, NVR）18例，完全病毒学应答患者（complete virologicalresponse,CVR）32例，部分病毒学应答患者(partial virological response,PVR)20例，健康对照10例。 1体外干扰素-α孵育PBMCs：留取70例受试者治疗前新鲜外周静脉血，，密度梯度离心法分离PBMCs，无血清培养基悬浮沉淀细胞，分为两组：第一组空白对照组（不加任何因子）；第二组加入干扰素-α因子刺激孵育PBMCs,4h后收集细胞。 2对所有受试者应用干扰素-α抗病毒治疗随访观察，留取治疗0周、1周、12周、24周时新鲜外周血，密度梯度离心法分离收集PBMCs。 3Trizol法抽提外周血单个核细胞总RNA，应用Real-time PCR法检测USP18、STAT1mRNA的表达水平。 4RIPA法提取细胞总蛋白应用western blot法检测USP18、STAT1、P-STAT1蛋白的表达水平。结果： 1健康对照组外周血PBMCs中USP18的表达为阴性 2干扰素-α治疗过程外周血PBMCs中USP18mRNA水平变化 70例CHB患者在干扰素-α治疗过程中接受mRNA水平检测，将PBMCs中内参GAPDH相对表达量设定为1.00，与GAPDH表达量相比较，干扰素-α治疗前NVR组PBMCs中USP18的表达水平(0.073±0.026)较CVR组（0.043±0.023）及PVR组（0.044±0.022）高，差异有统计学意义（F=10.665，P0.01）；治疗1周时，各组PBMCs中USP18的表达水平明显升高，NVR组（0.098±0.023）、CVR组（0.071±0.027）、PVR组（0.074±0.027），NVR组表达水平最高，差异有统计学意义（F=6.781，P=0.002）；治疗12周及24周时，各组表达水平为NVR组（0.097±0.024）(0.096±0.023)、CVR组（0.069±0.025）（0.066±0.025）、PVR组（0.075±0.026）（0.074±0.025），NVR组表达水平仍最高，差异有统计学意义（F1=7.140，p=0.002；F2=8.654, P0.01）。 3干扰素-α治疗过程外周血PBMCs中USP18蛋白水平变化将PBMCs中内参β-actin相对表达量设定为1.00，与β-actin表达量相比较，干扰素-α治疗前NVR组外周血PBMCs中USP18的表达水平(0.308±0.046)较CVR组（0.250±0.044）及PVR组（0.248±0.048）高，差异有统计学意义（F=11.151，P0.01）；治疗1周后，各组USP18的表达水平明显升高，NVR组（0.563±0.050）、CVR组（0.431±0.047）、PVR组（0.428±0.047），NVR组表达水平最高，差异有统计学意义（F=51.722，P0.01）；治疗12周及24周时，各组表达水平为NVR组（0.568±0.051）(0.566±0.049)、CVR组（0.430±0.047）（0.429±0.045）、PVR组（0.426±0.047）（0.430±0.049），NVR组表达水平仍最高，差异有统计学意义（F1=55.958，P0.01；F2=55.841, P0.01）。 4体外干扰素-α孵育刺激PBMCs后形态学观察 PBMCs经2h贴壁后，可见部分细胞悬浮，多数细胞为B细胞和外周血DC，干扰素-α孵育刺激4h后未见形态学变化，细胞数目无明显变化。 5体外干扰素-α刺激PBMCs后USP18、STAT-1Mrna/蛋白水平变化体外干扰素-α刺激治疗前外周血PBMCs后，干扰素-α组PBMCs中USP18在mRNA/蛋白上的表达水平高于空白对照组，差异有统计学意义（P0.01），而NVR组、CVR组及PVR组外周血PBMCs中STAT-1mRNA及其蛋白水平无显著性差异。 6体外干扰素-α刺激PBMCs后USP18表达水平与P-STAT-1表达水平的相关性空白对照组及干扰素-α组PBMCs中P-STAT-1表达水平分别为：NVR组（0.25±0.02）、（0.58±0.05），CVR组（0.26±0.02）、（0.66±0.05），PVR组（0.26±0.01）、（0.65±0.05），干扰素-α组PBMCs中P-STAT-1表达水平高于空白对照组，差异有统计学意义（P0.01），回归分析结果显示：干扰素-α刺激后PBMCs中USP18的表达水平与P-STAT-1的表达水平成负相关性，相关系数为：-0.753（P0.01）。 7多种影响因素与干扰素-α治疗疗效的回归分析将受试者的年龄、治疗前HBV-DNA基线水平、肝组织炎症分级、治疗前ALT水平、及治疗前PBMCs中USP18的表达水平多种因素与干扰素-α治疗疗效进行logisitic回归分析，通过逐步排除法，病毒载量、肝组织炎症分级及治疗前PBMCs中USP18的表达水平进入回归方程，y=0.588+20.552x1-39.299x2+19.641x3，回归方程有统计学意义（χ~2=35.682，P0.01）。结论： 1USP18的表达受干扰素刺激机体强烈诱导。 2USP18通过干扰STAT1的磷酸化作用，抑制干扰素-α抗病毒信号通路。 3HBV-DNA基线水平、肝脏炎症分级及治疗前PBMCs中USP18表达水平与干扰素-α治疗疗效均存在相关性，受试者治疗前PBMCs中高水平表达USP18影响干扰素-α治疗疗效。 4USP18可能是干扰素-α治疗慢乙肝过程中一个重要的稳定的负性预测因子。
[Abstract]:Objective: hepatitis B virus (HBV) infection has become a global public health problem, about 1 million people die of liver diseases associated with HBV infection every year. Active HBV replication is the key factor in liver injury and disease progression. So the maximum long-term inhibition or removal of HBV is the overall goal of chronic hepatitis B treatment. There are two major categories of drugs used for antiviral treatment of chronic hepatitis B: 1, nucleoside (acid) analogues, 2, interferon (IFN). Interferon has the dual effects of antiviral and immunoregulation, the treatment course is short and the drug resistance is rare, so it has gradually become the main choice for the treatment of chronic hepatitis B in the clinic. However, interferon is currently used. The treatment can only reach 30%-40% HBeAg serological conversion, about 60% of the patients still suffer from hepatitis B virus, although they accept interferon antiviral therapy, so it is particularly important to predict the therapeutic effect of interferon in the short term. The latest research focus is interferon stimulating gene in the interferon's antiviral signaling pathway, and ubiquitin specific Sex protease 18 (Ubiquitin-specific protease18, USPl8) is one of the important interferon stimulating genes. This study aims to explore the predictive significance and mechanism of USPl8 in the treatment of chronic hepatitis B by interferon - alpha.
Methods: 70 patients with chronic HBV infection were selected for all patients with immune clearance. All the patients were performed liver biopsy and no contraindication to interferon antiviral therapy. PBMCs was extracted before treatment with the extracorporeal interferon alpha incubation test, and the subjects were treated with interferon alpha antiviral therapy. For 24 weeks, there were 18 cases of nonvirological response (NVR), 32 cases of fully virological response (complete virologicalresponse, CVR), 20 cases of virological response (partial virological response, PVR), and 10 healthy controls.
1 in vitro interferon - alpha incubation PBMCs: 70 cases of fresh peripheral venous blood were left before treatment, PBMCs was separated by density gradient centrifugation, and serum free medium suspension cells were divided into two groups: the first group blank control group (without any factors); the second group added interferon alpha factor to incubate PBMCs, and then collect cells after 4H.
2 all subjects were followed up with interferon - alpha antiviral therapy, and the treatment was 0 weeks, 1 weeks, 12 weeks, and 24 weeks of fresh peripheral blood, and the density gradient centrifugation was used to collect PBMCs..
3Trizol method was used to extract the total RNA of peripheral blood mononuclear cells, and the expression level of USP18 and STAT1mRNA was detected by Real-time PCR.
4RIPA method was used to extract the total protein of the cell. The expression level of USP18, STAT1 and P-STAT1 protein was detected by Western blot.
Result:
1 the expression of USP18 in peripheral blood PBMCs was negative in healthy controls.
Changes of USP18mRNA levels in peripheral blood PBMCs during treatment with interferon alpha 2
70 cases of CHB patients received mRNA level detection during interferon alpha therapy, and the relative expression of GAPDH in PBMCs was set to 1. Compared with GAPDH expression, the expression level of USP18 in NVR group before interferon alpha therapy (0.073 + 0.026) was higher than that in the CVR group (0.043 + 0.023) and PVR group (0.044 + 0.022), and the difference was statistically significant (F=10.665, P). 0.01); at the 1 week of treatment, the expression level of USP18 in each group of PBMCs increased significantly, in group NVR (0.098 + 0.023), in group CVR (0.071 + 0.027), in group PVR (0.074 + 0.027), and in group NVR, the difference was statistically significant (F=6.781, P=0.002), and the expression level of each group was NVR (0.097 0.024) (0.096 + 0.023) at 12 and 24 weeks, CVR group. (0.066 + 0.025), group PVR (0.075 + 0.026) (0.074 + 0.025), and NVR group had the highest level of expression (F1=7.140, p=0.002, F2=8.654, P0.01).
Changes of USP18 protein level in peripheral blood PBMCs during treatment with interferon alpha 3
The relative expression of beta -actin in PBMCs was set to 1. Compared with the expression of beta -actin, the expression level of USP18 in PBMCs in peripheral blood of NVR group before interferon alpha therapy was higher than that in CVR group (0.250 + 0.044) and PVR group (0.248 + 0.048), and the difference was statistically significant (F=11.151, P0.01). After 1 weeks, the expression level of USP18 in each group was clear. In group NVR (0.563 + 0.050), group CVR (0.431 + 0.047), group PVR (0.428 + 0.047), the expression level of group NVR was the highest, the difference was statistically significant (F=51.722, P0.01). At 12 and 24 weeks, the expression level of each group was NVR (0.568 + 0.051) (0.566 + 0.049), CVR group (0.430 + +), NVR group table The level reached the highest level, and the difference was statistically significant (F1=55.958, P0.01; F2=55.841, P0.01).
4 morphological observation of PBMCs stimulated by interferon alpha in vitro
After the PBMCs was adhered to the 2H, some cells were suspended. Most of the cells were B cells and peripheral blood DC. No morphological changes were observed after interferon - alpha was incubated to stimulate 4h, and there was no significant change in the number of cells.
5 changes of USP18 and STAT-1Mrna/ protein levels after stimulation of PBMCs with interferon alpha in vitro
The expression level of USP18 on mRNA/ protein in the interferon - Alpha Group PBMCs was higher than that in the blank control group after interferin - alpha stimulation in the peripheral blood PBMCs. The difference was statistically significant (P0.01), but there was no significant difference in the level of STAT-1mRNA and protein in PBMCs in the group of NVR, CVR and PVR.
6 the correlation between USP18 expression and P-STAT-1 expression after PBMCs stimulation in vitro
The expression level of P-STAT-1 in the blank control group and the interferon - Alpha Group PBMCs were: group NVR (0.25 + 0.02), (0.58 + 0.05), group CVR (0.26 + 0.02), (0.66 + 0.05), PVR group (0.26 + 0.01), (0.65 + 0.05), P-STAT-1 in interferon Alpha Group PBMCs higher than that of blank control group, the difference was statistically significant (P0.01), and the regression analysis showed interference. After PBMCs stimulation, the expression level of USP18 in P-STAT-1 was negatively correlated with the expression level of P-STAT-1, and the correlation coefficient was -0.753 (P0.01).
Regression analysis of 7 factors and efficacy of interferon alpha therapy
The subjects' age, pre treatment HBV-DNA baseline level, liver tissue inflammation classification, pre treatment ALT level, and USP18 expression levels in PBMCs before treatment were analyzed by logisitic regression analysis with interferon - alpha therapy, by stepwise exclusion, viral load, liver tissue inflammation classification and the expression level of USP18 in PBMCs before treatment. Regression equation, y=0.588+20.552x1-39.299x2+19.641x3, regression equation has statistical significance (chi ~2=35.682, P0.01).
Conclusion:
The expression of 1USP18 is strongly induced by interferon.
2USP18 inhibits interferon alpha antiviral signaling pathway by interfering with phosphorylation of STAT1.
3HBV-DNA baseline level, liver inflammation classification and the level of USP18 expression in PBMCs before treatment are correlated with interferon - alpha therapy, and high level expression of USP18 in PBMCs before treatment affects the therapeutic effect of interferon - alpha.
4USP18 may be an important and stable negative predictor of interferon alpha in the treatment of chronic hepatitis B.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.62

【共引文献】